Optimization of Sunflower Helianthus Annus L Chloroplast Transformation and Transgene Expression

Abstract

Successful engineering of chloroplast genome in 1990s provided a significant breakthrough for genetic improvement in plants. Chloroplast is proved to be an ideal site for production of recombinant proteins of both therapeutic and agronomic importance. Through our study which was based upon optimization of sunflower chloroplast transformation and transgene expression, we could achieve expression of transgene (gfp) in transformed sunflower callus. newlineAs per the proposed objectives, optimization of direct regeneration of sunflower plantlets from primordial meristem / shoot tip explants was achieved on MS media containing BAP (4.44 and#956;M L-1) and NAA (2.69 and#956;M L-1). Regeneration of hypocotyl explants was achieved on MS media containing BAP (2.22 and#956;M L-1) alone. In case of leaf explants, regeneration was not achieved but they could be induced for callus formation on two best combinations of MS media; one containing BAP (2.22 and#956;M L-1), NAA (0.54 and#956;M L-1), GA3 (0.29 and#956;M L-1), 20 % de-proteinized coconut water and AgNO3 (10 and#956;M) while other with BAP (8.88 and#956;M L-1) and NAA (2.69 and#956;M L-1). newlineSimilarly, cotyledon regeneration using available report in literature, regeneration in cotyledons explants (of 48 hrs age) was achieved as per our laboratory conditions with some modifications. To summarize, cotyledons were initiated for shoot regeneration on MS with 6-BAP (8.88 and#956;M L-1), IAA (5.71 and#956;M L-1). Initiated shoots were elongated on MS with BAP (2.22 and#956;M L-1), GA3 (0.29 and#956;M L-1). Elongated shoots were initiated for root formation on half MS with NAA (2.69 and#956;M L-1). For chloroplast transformation, sunflower leaves and cotyledons (of 48 hrs age) were used as explants. newlineFor sunflower chloroplast transformation, a synthetic construct, pSFSRT (Plasmid Sunflower Swami Ramanand Teerth) was designed and constructed. This synthetic construct (length 5131 bp) was based on pUC57 backbone having flanking sequences: rbcL-accD, selectable marker: nptII, reporter gene: gfp; promoters: Prrn (for nptII), PpsbA (for gfp) and 5 and 3 UTRs (from psbA, rps16 genes).